Secreted AGR2 Helps Tumor Cells to Establish Its Microenvironment

Transcription

1 SJTU-School of Pharmacy Secreted AGR2 Helps Tumor Cells to Establish Its Microenvironment Dawei Li, Prof. Center For Cell Engineering And Antibody Medicine Shanghai Jiao Tong University 1

2 Tumor Microenvironment: Multiple Cell Types, Factors, Receptors Growth Factors VEGF bfgf EGF etc Immuno Regulation Proteases Morphogens 2

3 AGR2 Is a Key Factor in Tissue Remodeling AGR2 is critical in regeneration and promotes cancer formation Science (2007) Kumar et al 3

4 Non-tumor Cells: Targeting Cancer Microenvironment: Beyond Angiogenesis? Tumor Remodels Its Microenvironment With Multiple Secreted Factors Migration Cell Cycle Vascularization 4

5 AGR2 Is a Drug Target Against Cancer AGR2 Overexpression increases tumorigenic potential Cancer Research (2008) Wang Zheng et al 5

6 AGR2 Is Expressed Through Low Serum Induction Surface AGR2 Internal AGR2 6

7 Angiostatic Effect of Reduce HUVE Cell Tube Formation Degree of HUVC tube formation Anti-VEGF Control Anti VEGF Anti AGR2 Anti-AGR2 7

8 AGR2 Is A Multi-Functional Proliferation Enhancer Control AGR2 FGF FGF+AGR2 8

9 荧光 Confocal 共定位 -AGR2 和 bfgf 相互作用 Binding with bfgf, then binding to the membrane of HUVEC.

10 CAGR2 Has No Effect on Ang-1/PLGF/PDGF Induced Migration 0.5% serum AGR2 50ng/ml Ang-1 50ng/ml PLGF 50ng/ml PDGF 25ng/ml Ang-1 50ng/ml AGR2 50ng/ml PLGF 50ng/ml AGR2 50ng/ml PDGF 50ng/ml AGR2 50ng/ml

11 AGR2 增强 bfgf / VEGF 在细胞转移实验中的作用 对照 AGR2 VEGF VEGF+AGR2 对照 AGR2 bfgf AGR2+bFGF

12 AGR2 can enhance the phosphorylation of ERK and VEGFR caused by VEGF and this effection can be disturbed by VEGFR inhibitor or VEGF antibody Axitinib 2nM PD nM Avastin 0 AGR2 VEGF AGR2 VEGF AGR2 VEGF AGR2 VEGF AGR2 AGR2 VEGF VEGF VEGF VEGF perk ERK actin 0 AGR2 VEGF 3ng/ml VEGF 20ng/ml AGR2 AGR2 pvegfr (1175) perk actin

13 DTT can disturb the AGR2 signaling activities DTT 250nM 250nM 5uM 5uM 100uM 100uM 0 VEGF AGR2 VEGF AGR2 VEGF AGR2 VEGF AGR2 VEGF VEGF VEGF VEGF perk ACTIN AGR2 抗体阻断 VEGF 对 ERK1/2 的促进作用 0 AGR2 VEGF AGR2 清除 AGR2 中和 AGR2 VEGF AGR2 VEGF AGR2 AGR2 AGR2 VEGF AGR2 VEGF VEGF perk actin

14 Generation of Agtuzumab Against AGR2 Hybridoma: Patent #ZL Sequence and Target: Patent Pending Agtuzumab: International Patent -PCT and Countries AGR2 is an early marker for ovarian cancer Agtuzumab from A specifically binds AGR2 in its denatured or native form Agtuzumab specifically inhibits AGR2 s growth enhancing function 14

15 AGR2 mab Inhibits Cancer Cell Growth A B PBS Tumor volume (mm3) PBS Number of days after implantation D 1 * C PBS Tumor weight (g) PBS A. mab inhibit the growth of SKOV3 Xenograft model of nude mice. Comparison of tumor size between control group and treatment group. B. Growth curve of the SKOV3 allograft tumors of nude mice. C and D, Comparison of tumor size and weight. 15

16 The Angiostatic Effect 8A4 Inhibits cancer cell growth in vivo A B Human-AGR2 SKOV3 Tumor Human-GAPDH C D Control Tumor - + SKOV3 AGR2 GAPDH PBS E Detection of AGR2 expression by semi-quantitative RT-PCR AG GR2/GAPDH SKOV3 cell culture SKOV3 Xenograft tumor PBS Ct m-vegfa m-vegfr2 Ct h-vegfa h-vegfr2 A. B, PCR of the cdna of 0.5ml tumor tissue with 25 cycles shows that human AGR2 is expressed in tumors, but SKOV3 doesn t express AGR2. AGR2 decrease in tumor tissue of treatment group. C, expression of AGR2 in SKOV3 cell and SKOV3 xenograft tumor. D E, Figure 6. Comparison of the expression of mouse and humon 16 growth factors in tumor by realtime PCR.

17 Angiostatic Effect Reduced Angiogenesis in Tumor Tissue After Treatment Control Tumor Section Stained with CD31, a Blood vessel marker Control 17

18 AGR2 Enhances Growth Factor Effect Cancer Cells AGR2 Gradient AGR2 EGF/EGFR Chemotactic effect Stroma cells Blood vessels Other cells FGF/FGFR VEGF/VEGFR 18

19 ACKNOWLEDGEMENT Lab members: Zhenghua Wu Weiguo Xu Qi Zhu Guangwei Gao Hao Guo Dhairi Mashuasi Hao Chen Haochuan Lou Collaborators Dr. Chuanhua He (Yale/Int. Med) Dr. Ronny Drapkin (Harvard/DFCI) Dr. Steven Skates (Harvard/MGH) 19